Our work on tRNA biosynthesis shows that tRNA molecules are transcribed into large precursor RNA molecules which are converted into tRNA through a highly ordered series of steps catalyzed by specific enzymes. It is observed that in cells which lack a specific enzyme, the appearance of tRNA is blocked, and the substrate of the missing enzyme accumulates. This analysis of the nucleotide sequence of the accumulated precursor RNA then demonstrates which enzymatic alterations have occurred in previous steps. This kind of approach coupled with the availability of a mutant in each enzyme has recently allowed us to propose five steps by which one specific precursor RNA is converted into tRNA. We are now confirming this pathway by performing the entire five-step process in vitro using purified enzymes and precursor RNA intermediates. Finally, attempts to define the nucleotide residues in a precursor RNA that are responsible for the accurate and faithful enzymatic recognition of precursor RNA during its conversion into tRNA. These studies involve the isolation of a series of mutationally altered precursor RNAs that are blocked in the production tRNA, determining the nucleotide location of each mutation, and correlating this data with the effects of the mutation on the enzymatic processing of precursor RNA. BIBLIOGRAPHIC REFERENCES: Seidman, J.G. & McClain, W.H., 1975. Three Steps in Conversion of Large Precursor RNA into Serine and Proline Transfer RNAs. Proc. Nat. Acad. Sci. USA 72: 1491-1495. Seidman, J.G., Schmidt, F.J., Foss, K. & McClain, W.H., 1975. A Mutant of Escherichia coli Defective in Removing 3 feet Terminal Nucleotides from Some Transfer RNA Precursor Molecules. Cell 5: 389-400.